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ICAMs redistributed by chemokines to cellular uropods as a mechanism for recruitment of T lymphocytes.

del Pozo MA, Cabañas C, Montoya MC, Ager A, Sánchez-Mateos P, Sánchez-Madrid F - J. Cell Biol. (1997)

Bottom Line: Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment.Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process.An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Immunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain.

ABSTRACT
The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte-endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.

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Freshly isolated CD45RO+ lymphocytes are able to recruit cells through the uropod. (A and B) Measurement of cell recruitment mediated by cell uropods. The total population of PBL  (A), CD45RA+, or CD45RO+ cells (B) were allowed to bind to  plastic petri dishes coated with ICAM-1–Fc for 30 min at 37°C in  the presence of medium alone, 10 ng/ml RANTES, MCP-1, MIP1α, MIP-1β, or IL-8. After addition of a second cohort of PBL  from the same donor, cell–cell interactions were recorded for 1 h,  and the recruitment index was estimated as described in Materials and Methods. Arithmetic mean ± 1 SD of four independent  experiments performed with PBL (A) or two independent ones  with CD45RA+/CD45RO+ cells (B) is shown. (C) Freshly isolated CD45RA+ (a and c) or CD45RO+ (b and d) cells were allowed to bind to coverslips coated with 10 μg/ml ICAM-1–Fc for  30 min at 37°C in the presence (c and d) or in the absence (a and  b) of 10 ng/ml MIP-1α. Fixed cells were then stained for ICAM-3  with TP1/24 mAb, and the proportion of uropod-bearing cells  was calculated as described in Materials and Methods. The percentage of polarized cells in CD45RA+ versus CD45RO+ cells  was as follows: untreated 5 vs 19%, RANTES 11 vs 29%, MCP-1  10 vs 32%, MIP-1α 3 vs 26%, MIP-1β 4 vs 22%, and IL-8 8 vs  20%. The cellular uropod and the cell contact area with the substratum were indeed in a distinct plane of focus.
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Figure 4: Freshly isolated CD45RO+ lymphocytes are able to recruit cells through the uropod. (A and B) Measurement of cell recruitment mediated by cell uropods. The total population of PBL (A), CD45RA+, or CD45RO+ cells (B) were allowed to bind to plastic petri dishes coated with ICAM-1–Fc for 30 min at 37°C in the presence of medium alone, 10 ng/ml RANTES, MCP-1, MIP1α, MIP-1β, or IL-8. After addition of a second cohort of PBL from the same donor, cell–cell interactions were recorded for 1 h, and the recruitment index was estimated as described in Materials and Methods. Arithmetic mean ± 1 SD of four independent experiments performed with PBL (A) or two independent ones with CD45RA+/CD45RO+ cells (B) is shown. (C) Freshly isolated CD45RA+ (a and c) or CD45RO+ (b and d) cells were allowed to bind to coverslips coated with 10 μg/ml ICAM-1–Fc for 30 min at 37°C in the presence (c and d) or in the absence (a and b) of 10 ng/ml MIP-1α. Fixed cells were then stained for ICAM-3 with TP1/24 mAb, and the proportion of uropod-bearing cells was calculated as described in Materials and Methods. The percentage of polarized cells in CD45RA+ versus CD45RO+ cells was as follows: untreated 5 vs 19%, RANTES 11 vs 29%, MCP-1 10 vs 32%, MIP-1α 3 vs 26%, MIP-1β 4 vs 22%, and IL-8 8 vs 20%. The cellular uropod and the cell contact area with the substratum were indeed in a distinct plane of focus.

Mentions: Although T lymphoblasts likely represent the memoryactivated subset of T cells, the number of circulating lymphoblasts is very low. Therefore, we explored whether the mechanism of cell recruitment mediated by uropods could be observed in lymphocytes directly isolated from the blood. Although these cells adhered nicely onto the ICAM-1– coated surface, they moved less than activated T lymphoblasts, and chemokines only weakly induced lymphocyte polarization and recruitment of a second layer of unstimulated peripheral blood lymphocytes (Fig. 4 A, and data not shown). Therefore, we decided to explore whether the recruitment was selectively exerted by a subset of the cells. In this regard, it has recently been reported that most of the chemokine receptors are upregulated on the CD45R0+ memory-activated phenotype cells (Loetscher et al., 1996; Mackay, 1996; Qin et al., 1996). Thus, we isolated CD45RA+ naive and CD45R0+ memory peripheral blood lymphocytes and compared their ability to contact, engage, and drive other lymphocytes. Chemokine-mediated T cell recruitment was much more efficiently promoted by CD45R0+ cells than by CD45RA+ naive cells (Fig. 4 B). Accordingly, CD45R0+ showed a higher polarized morphology, both in basal conditions and after chemokine treatment (Fig. 4 C, b and d). CD45R0+ cells developed a proper uropod, and they actively locomote on the ICAM-1–coated surface, whereas the CD45RA+ cells only displayed a capping of the ICAM-3 molecule and migrated slower (Fig. 4 C and data not shown). These data indicate that CD45R0+ memory T cells responded more efficiently to chemokines likely due to the higher expression of chemokine receptors on these cells and therefore can account for the T cell recruitment observed in the total population of freshly isolated lymphocytes.


ICAMs redistributed by chemokines to cellular uropods as a mechanism for recruitment of T lymphocytes.

del Pozo MA, Cabañas C, Montoya MC, Ager A, Sánchez-Mateos P, Sánchez-Madrid F - J. Cell Biol. (1997)

Freshly isolated CD45RO+ lymphocytes are able to recruit cells through the uropod. (A and B) Measurement of cell recruitment mediated by cell uropods. The total population of PBL  (A), CD45RA+, or CD45RO+ cells (B) were allowed to bind to  plastic petri dishes coated with ICAM-1–Fc for 30 min at 37°C in  the presence of medium alone, 10 ng/ml RANTES, MCP-1, MIP1α, MIP-1β, or IL-8. After addition of a second cohort of PBL  from the same donor, cell–cell interactions were recorded for 1 h,  and the recruitment index was estimated as described in Materials and Methods. Arithmetic mean ± 1 SD of four independent  experiments performed with PBL (A) or two independent ones  with CD45RA+/CD45RO+ cells (B) is shown. (C) Freshly isolated CD45RA+ (a and c) or CD45RO+ (b and d) cells were allowed to bind to coverslips coated with 10 μg/ml ICAM-1–Fc for  30 min at 37°C in the presence (c and d) or in the absence (a and  b) of 10 ng/ml MIP-1α. Fixed cells were then stained for ICAM-3  with TP1/24 mAb, and the proportion of uropod-bearing cells  was calculated as described in Materials and Methods. The percentage of polarized cells in CD45RA+ versus CD45RO+ cells  was as follows: untreated 5 vs 19%, RANTES 11 vs 29%, MCP-1  10 vs 32%, MIP-1α 3 vs 26%, MIP-1β 4 vs 22%, and IL-8 8 vs  20%. The cellular uropod and the cell contact area with the substratum were indeed in a distinct plane of focus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139764&req=5

Figure 4: Freshly isolated CD45RO+ lymphocytes are able to recruit cells through the uropod. (A and B) Measurement of cell recruitment mediated by cell uropods. The total population of PBL (A), CD45RA+, or CD45RO+ cells (B) were allowed to bind to plastic petri dishes coated with ICAM-1–Fc for 30 min at 37°C in the presence of medium alone, 10 ng/ml RANTES, MCP-1, MIP1α, MIP-1β, or IL-8. After addition of a second cohort of PBL from the same donor, cell–cell interactions were recorded for 1 h, and the recruitment index was estimated as described in Materials and Methods. Arithmetic mean ± 1 SD of four independent experiments performed with PBL (A) or two independent ones with CD45RA+/CD45RO+ cells (B) is shown. (C) Freshly isolated CD45RA+ (a and c) or CD45RO+ (b and d) cells were allowed to bind to coverslips coated with 10 μg/ml ICAM-1–Fc for 30 min at 37°C in the presence (c and d) or in the absence (a and b) of 10 ng/ml MIP-1α. Fixed cells were then stained for ICAM-3 with TP1/24 mAb, and the proportion of uropod-bearing cells was calculated as described in Materials and Methods. The percentage of polarized cells in CD45RA+ versus CD45RO+ cells was as follows: untreated 5 vs 19%, RANTES 11 vs 29%, MCP-1 10 vs 32%, MIP-1α 3 vs 26%, MIP-1β 4 vs 22%, and IL-8 8 vs 20%. The cellular uropod and the cell contact area with the substratum were indeed in a distinct plane of focus.
Mentions: Although T lymphoblasts likely represent the memoryactivated subset of T cells, the number of circulating lymphoblasts is very low. Therefore, we explored whether the mechanism of cell recruitment mediated by uropods could be observed in lymphocytes directly isolated from the blood. Although these cells adhered nicely onto the ICAM-1– coated surface, they moved less than activated T lymphoblasts, and chemokines only weakly induced lymphocyte polarization and recruitment of a second layer of unstimulated peripheral blood lymphocytes (Fig. 4 A, and data not shown). Therefore, we decided to explore whether the recruitment was selectively exerted by a subset of the cells. In this regard, it has recently been reported that most of the chemokine receptors are upregulated on the CD45R0+ memory-activated phenotype cells (Loetscher et al., 1996; Mackay, 1996; Qin et al., 1996). Thus, we isolated CD45RA+ naive and CD45R0+ memory peripheral blood lymphocytes and compared their ability to contact, engage, and drive other lymphocytes. Chemokine-mediated T cell recruitment was much more efficiently promoted by CD45R0+ cells than by CD45RA+ naive cells (Fig. 4 B). Accordingly, CD45R0+ showed a higher polarized morphology, both in basal conditions and after chemokine treatment (Fig. 4 C, b and d). CD45R0+ cells developed a proper uropod, and they actively locomote on the ICAM-1–coated surface, whereas the CD45RA+ cells only displayed a capping of the ICAM-3 molecule and migrated slower (Fig. 4 C and data not shown). These data indicate that CD45R0+ memory T cells responded more efficiently to chemokines likely due to the higher expression of chemokine receptors on these cells and therefore can account for the T cell recruitment observed in the total population of freshly isolated lymphocytes.

Bottom Line: Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment.Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process.An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Immunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain.

ABSTRACT
The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte-endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.

Show MeSH
Related in: MedlinePlus